WP744 is a novel anthracycline with enhanced activity against neuroblastoma1

Journal of Surgical Research 121, 187–196 (2004) doi:10.1016/j.jss.2004.03.027

WP744 Is a Novel Anthracycline with Enhanced Activity Against Neuroblastoma 1 Thomas H. Inge, M.D., Ph.D.,*,†,2 Nathaniel L. Harris, B.S.,† Jianqiang Wu, M.D.,† Richard G. Azizkhan, M.D.,*,† and Waldemar Priebe, Ph.D.‡ *Children’s Hospital Research Foundation and Department of Pediatric Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; †Department of Surgery, University of Cincinnati, Cincinnati, OH; and ‡The University of Texas M.D. Anderson Cancer Center, Houston, TX Submitted for publication January 10, 2004

Background. Doxorubicin (Dox) is one of the most useful chemotherapeutic agents for patients with advanced neuroblastoma (NB). A series of Dox analogs with bulky substitutions at the C-4= at amino-sugar have been designed to impair interactions between the drug and P-glycoprotein (P-gp), a multidrug drug resistance (MDR) transporter. Two analogs, WP744 and WP769, were selected and their biological properties were compared with Dox and the daunorubicinbased bisintercalator WP631. These novel Dox analogs may have antitumor activity beyond MDR evasion. Materials and methods. MTT assays were used to determine the potency of three structurally altered Dox analogs against a panel of NB cell lines with and without amplification of the MYCN oncogene. Flow cytometry (FCM) was used to analyze apoptosis and cell death and phenotype cell lines for surface expression of the MDR protein P-gp. Results. The 4=-O-benzylated Dox analogs WP744 and WP769 were 2 to 36 times more cytotoxic than Dox for the NB cell lines tested. The bis-intercalator WP631, despite its significantly greater affinity for DNA (>10,000-fold), was generally less potent against NB than Dox. In Tet21N cells, which conditionally express MYCN, greatly enhanced (nearly 6-fold) sensitivity to WP744 killing was seen when this oncogene was induced, while enhanced sensitivity to Dox was more modest (2-fold) under MYCN-induced conditions. Treatment with WP744 also resulted in enhanced apoptosis. Apoptosis, but not cell death, in response to either WP744 or Dox was inhibited by caspase inhibi1 This research was supported by a Translational Research Award from the Cincinnati Children’s Hospital Medical Center to T.I. 2 To whom correspondence and reprint requests should be addressed at Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail: [email protected]

tion, suggesting that cell death was not completely dependent upon apoptosis. P-gp expression was detectable on five NB cell lines. WP744 was more cytotoxic than Dox against both P-gp ⴙ and P-gp ⴚ cells. Conclusions. These findings demonstrate that 4=-Obenzylation of the anthracycline molecule significantly enhances potency against NB independent of MYCN status, caspase activation, and MDR phenotype. However, WP744 demonstrated a unique synergy with MYCN for cell killing when this oncogene was specifically induced. WP744 may be more useful than conventional agents for the treatment of tumor clones that harbor defects in apoptotic pathways, in those with MYCN amplification, and in those with drug-resistant tumors. © 2004 Elsevier Inc. All rights reserved. Key Words: neuroblastoma; chemotherapy; doxorubicin; anthracycline; apoptosis; cell death; WP744; WP769; WP631. INTRODUCTION

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and is therefore one of the most common tumors seen by pediatric surgeons [1]. Despite aggressive surgical resections and numerous potent chemotherapeutic agents, most patients with advanced NB still succumb to disease. Over the past several decades, improvements in our understanding of tumor biology have enabled the rational development of novel antineoplastic agents and regimens, and clinical outcomes for some pediatric malignancies such as germ cell tumors, lymphomas, and nephroblastomas have improved [2]. However, despite major research efforts, only incremental advances in treatment of NB have been made [3]. Doxorubicin (Dox) is an anthracycline antibiotic that

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remains an important agent in the multidrug regimens for NB as well as many epithelial malignancies of adulthood and childhood. Since the discovery of anthracyclines over 30 years ago, investigators have sought to determine what specific features of the Dox molecule account for the broad-range anticancer properties of this drug. To date, many analogs have been synthesized in search of more potent antineoplastic agents with fewer adverse effects. For most anthracycline-sensitive solid tumors like NB, Dox remains the anthracycline of choice. Doxorubicin and related anthracyclines are topoisomerase II poisons that intercalate into DNA, can form free radicals, trigger apoptosis associated with mitochondrial cytochrome release, and have direct cell membrane toxicity [4 –10]. Despite pleiotropic mechanisms of action resulting in potent anti-tumor effects, MDR frequently develops [11]. The most studied forms of MDR in NB are mediated by overexpression of the MDR1 gene that encodes the plasma membrane P-glycoprotein (P-gp) [12–16]. P-gp molecules are responsible for removing toxic molecules from normal cells, while some tumor cells have clearly exploited this adaptive mechanism and can withstand higher concentrations of drug treatment. This molecule mediates resistance against a broad range of drugs including Dox [17, 18]. Analogs of Dox have been designed with substitutions to alter functionality to bypass drug resistance [19, 20]. The most profound changes in anti-tumor activity of the anthracycline have been seen after chemical alteration of the amino sugar [21, 22]. Analogs have been developed that retain anti-tumor effects of Dox but lack recognition by P-gp [23–25]. A unique class of analogs is represented by WP631 (Fig. 1), a bis-anthracycline that blocks Sp1activated transcription [26] and also blocks Sp-1 interaction within the MYCN promoter [27]. WP744 and WP769 are novel anthracyclines which are benzylated at the C=-4 hydroxyl of Dox. The aim of the present study was to compare the cytotoxic activity of novel Dox analogs against NB and assess the mechanism by which cell death occurred following drug treatment. Our results show that the O-benzylated anthracyclines were cytotoxic against all NB cell lines tested. Although classic features of apoptosis were seen, cell death was not dependent upon apoptosis. MATERIALS AND METHODS Drugs and Reagents WP631, WP769, and WP744 were synthesized in the laboratory of one of the co-authors (W.P.; see Fig. 1). WP769 is identical to WP744 with the exception of an additional hydroxyl group to the D ring of the aglycone. Dox was purchased from Sigma-Aldrich Corp. (Milwaukee, WI). Stock solutions of anthracyclines were prepared by dissolving the compounds in distilled water and storing at ⫺20°C. Propidium iodide (PI) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (Milwaukee, WI). The caspase inhibitor n-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone (Z-VAD-

FMK) was obtained from Alexis Biochemicals (San Diego, CA). A stock solution (20 mM) was prepared by dissolving Z-VAD-fmk in sterile DMSO and stored at ⫺20°C until use.

Cell Lines The IMR-32 cell line was originally obtained from the American Type Culture Collection (ATCC, Manassas, VA) and was provided for these studies by Dr. Timothy Cripe (Children’s Hospital Research Foundation, Cincinnati, OH). IMR-32 cells were cultured in complete medium (CM) consisting of minimum essential alpha medium (Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal bovine serum (FBS; Gibco) and antibiotics (100 U/ml penicillin G, 100 ␮g/ml streptomycin sulfate, and 250 ng/ml amphotericin B, all obtained from Gibco). LA-N-5 cells were a gift from Dr. Robert Seeger (Children’s Hospital of Los Angeles, Los Angeles, CA) and were cultured in CM consisting of RPMI 1640 containing 10% FBS and antibiotics. The SK-N-SH cell line was cultured in CM consisting of Eagle’s minimum essential medium plus 10% FBS and antibiotics. The SK-N-BE(2) and SH-SY5Y cell lines were also obtained from the ATCC. Both cell lines were cultured in CM consisting of a 1:1 mixture of Eagle’s minimum essential medium with non-essential amino acids and Ham’s F12 nutrient mix (Gibco) with 10% FBS and antibiotics. The SMS-SANS, CHLA-20, and CHLA-79 cell lines [28, 29] were gifts from Dr. C. Patrick Reynolds (Children’s Hospital of Los Angeles). The CHLA-20, and CHLA-79 cell lines were cultured in CM consisting of Iscove’s modified Dulbecco’s medium containing 20% FBS, 3 mM L-glutamine, 5 mg/ml each of insulin, transferrin, selenous acid (Gibco), and antibiotics. The SMS-SANS cell line was cultured in CM consisting of RPMI 1640 media supplemented with 10% FBS and antibiotics. SHEP and subclones harboring vector alone (Tet2) or ectopically overexpressing human MYCN (Tet21N) were generous gifts from Dr. Manfred Schwab (German Cancer Research Center, Heidelberg, Germany). Their derivation has been described previously [30]. These cells were cultured in CM consisting of RPMI 1640 containing 10% FBS and antibiotics (100 U/ml penicillin G, 100 ␮g/ml streptomycin sulfate, and 250 ng/ml amphotericin B, Gibco). Expression of the extrachromosomal MYCN construct was conditionally regulated by the addition or removal of 1 ␮g/ml tetracycline; MYCN expression is suppressed by addition of tetracycline, while the removal of tetracycline results in abundant expression of MYCN. For routine passage every three to five days and prior to experimentation, cell lines were harvested using 0.05% trypsin and 0.02% EDTA without Ca 2⫹ and Mg 2⫹ (Gibco). All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Proliferation and Cell Viability Assays After drug treatment and incubation, viable cell density was assessed by the conversion of MTT to a formazan product [31]. NB cells were seeded at a density of 10 4 cells in each well of a 96-well flat-bottomed tissue culture plate (Falcon, Becton–Dickson Labware, Franklin Lakes, NJ) in 100 ␮l of complete medium with or without anthracyclines. Forty-eight hours following treatment, MTT was added to each well to a final concentration of 0.25 mg/ml and then incubated for 2 h at 37°C in the dark. The reaction was terminated by addition of 0.04 N HCl in isopropanol. Following thorough mixing to dissolve the formazan product, the plates were read at 570 nm on a micro ELISA plate reader (SpectraMax Plus, Molecular Devices, Sunnyvale, CA). Quadruplicate wells were used for each treatment group. Spectrophotometric data were analyzed by fundamental nonlinear regression to determine the concentration of drug resulting in 50% growth inhibition (IC 50) using PharmTools Pro computer software (The McCary Group, Elkins Park, PA). The arithmetic mean of IC 50 data from multiple experiments was obtained and statistical differences between treatment groups were assessed by Student’s t test.

INGE ET AL.: ENHANCED KILLING OF NEUROBLASTOMA WITH WP744

FIG. 1.

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Molecular structures of (A) Dox; (B) WP744; (C) WP769; and (D) WP631.

FCM Analysis for Cell Death, P-gp Expression, and Apoptosis Cells were plated at a density of 1 ⫻ 10 6 cells/well in 6-well plates and cultured overnight in complete medium with or without anthracyclines and harvested by trypsinization. To quantitate dead cells, adherent and non-adherent cells were collected, washed, and stained

with PI (5 ␮g/ml) for 15 min on ice prior to FCM analysis. Cellular debris was excluded based on forward- and side-angle light scatter characteristics. Cellular fluorescence was measured through a 585/ 42-band pass filter (FL-2) using log amplification to assess percentage of nonviable cells. To characterize P-gp expression, cells were harvested, washed in

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TABLE 1 NB Cell Line Characteristics NB line

Histology

IMR-32 LAN5 SK-N-BE(2) SH-SY5Y SK-N-SH SHEP SMS-SAN CHLA-20 CHLA-79

Neural Neural Neural Neural Schwannian ⫹ neural Schwannian

MYCN amplification

p53 wild-type

Yes Yes Yes No No No Yes No No

Yes Yes No Yes Yes Yes Yes Yes Yes

phenotyping solution (1⫻ PBS, 1% bovine serum albumin, 0.1% sodium azide), and incubated with anti-P-gp IgG 2a antibody (clone MRK-16; Kamiya Biomedical Co., Seattle, WA) for 30 min on ice, washed, and incubated with FITC-conjugated secondary antibody (anti-mouse IgG 2A/2B-FITC; BD PharMingen, San Diego, CA) for 30 min on ice. The MRK-16 antibody has been used by others to identify P-gp expression in NB [10]. Surface P-gp expression was assessed using FCM by monitoring FL1 intensity. A control group was stained with anti-mouse IgG 2A/2B-FITC without primary antibody to assess background fluorescence. Apoptosis was quantified by analysis of nuclear staining with PI after hypotonic lysis (0.1% sodium citrate, 0.01% Triton X-100, and 0.1 mg/ml propidium iodide; Sigma) as described [32]. For death and apoptosis assays, fluorescence data were collected on 10,000 events and analyzed using CellQuest software (Becton–Dickinson, San Diego, CA) on a microcomputer (Apple Corp., Cupertino, CA).

RESULTS Novel Anthracyclines Show Differential Activity Against NB

Clinically, NB tumors are made up of heterogeneous cell types; thus we initially chose to assess drug response using a heterogeneous panel of NB cell lines (Table 1). These cell lines have been used extensively in NB research and are heterogeneous with respect to their histology and MYCN amplification status. How-

ever, eight cell lines are wild-type for p53 expression. To determine whether structurally altered anthracyclines would be more cytotoxic to NB cells than the “parent” Dox molecule, in vitro MTT assays were performed. NB cells were treated with serial dilutions of anthracyclines (0 – 4 ␮M). After 4 – 6 days of culture, the MTT assay was used to determine the drug concentration resulting in IC 50. IMR-32, LAN5, SK-N-BE(2), and SHEP cells were 2- to 3-fold more sensitive to the O-benzylated anthracycline WP744 compared to Dox, although these differences were statistically significant only in IMR-32, SK-N-BE(2), and SHEP (Table 2). SH-SY5Y and SK-N-SH cells demonstrated 26- to 36fold enhanced sensitivity to WP744 compared to Dox (P ⬍ 0.05). There was generally excellent correlation between IC 50 findings with WP744 and the similar molecule WP769. The bis-anthracycline WP631 was less potent than Dox against most cell lines in this panel. NB cells are known to acquire a drug-resistant phenotype with increasing exposure to cytotoxic chemotherapy in vivo. Keshelava et al have described a series of cell lines—SMS-SAN, CHLA-20, and CHLA-79 — that were isolated from NB patients prior to therapy, after induction therapy, and after myeloablative therapy, respectively [29]. SMS-SAN is chemotherapy sensitive, while CHLA-20 and CHLA-79 cell lines express the MDR phenotype. We next sought to determine the sensitivity of these cells to novel anthracyclines. The SMS-SAN line was minimally more sensitive to WP744 than Dox (P ⬎ 0.05), while CHLA-20 cells were significantly more sensitive to WP744 than Dox (P ⬍ 0.05; Table 2). CHLA-79 cells were 6-fold more sensitive to WP744 than Dox (P ⬍ 0.001). Again, WP769 and WP744 demonstrated similar degrees of enhanced cytotoxicity against drug-resistant NB cells, while all three of these cell lines were markedly less sensitive to WP631 compared to Dox. Overall, these data demonstrate that compared to Dox, the novel O-benzylated anthracyclines were more cyto-

TABLE 2 Comparative Drug Potencies NB line

n

Dox IC 50

WP744 IC 50

WP769 IC 50

WP631 IC 50

IMR-32 LAN5 SK-N-BE(2) SHEP SH-SY5Y SK-N-SH SMS-SAN CHLA-20 CHLA-79

3 2 2 3 4 3 3 3 3

8⫾2 9 ⫾ 0.7 87 ⫾ 6 672 ⫾ 159 50 ⫾ 14 26 ⫾ 10 26 ⫾ 5 87 ⫾ 17 66 ⫾ 1

3 ⫾ 1* 4⫾2 29 ⫾ 9* 257 ⫾ 37* 1.4 ⫾ 1* 1 ⫾ 1* 17 ⫾ 6 26 ⫾ 2* 11 ⫾ 2*

4⫾2 3 ⫾ 0.1 28 ⫾ 5 381 ⫾ 146 1.7 ⫾ 1.3 25 ⫾ 16 12 ⫾ 2 35 ⫾ 15 13 ⫾ 2

10 ⫾ 1 18 ⫾ 2 26 ⫾ 6 181 ⫾ 33 170 ⫾ 25 55 ⫾ 7 38 ⫾ 4 366 ⫾ 106 727 ⫾ 305

Note. Regression analysis was performed on spectrophotometric data from quadruplicate wells at each serial drug dilution to determine the concentration of drug resulting in 50% inhibition (IC 50). The arithmetic mean IC 50 ⫾ SEM for “n” experiments is shown. Dox and WP744 treated groups were compared by Student’s t test and “*” indicates significantly different mean IC 50 values (P ⬍ 0.05).

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FIG. 2. MYCN expression in Tet-21/N cells after removal of tetracycline. Tet-21/N cells were treated with 1 ␮g/ml tetracycline for 1 week prior to washing and incubation in complete medium lacking tetracycline. Northern analysis for MYCN was performed immediately after washing (⫺), or at 6, 12, 18, 24, and 48 h of culture. These results are representative of result obtained in two such experiments.

481 nM). Strikingly, when MYCN expression was abundant, the sensitivity to WP744 was enhanced almost 6-fold (IC 50 ⫽ 12 nM versus 70 nM). To control for the potentially confounding effects of tetracycline treatment, identical experiments were performed with Tet2 control cells. As shown, no significant effect of tetracycline treatment was noted. These data suggest that WP744 significantly cooperates with the pro-apoptotic function of MYCN to enhance cell death in cultured NB cells. WP744 Induces Early Cell Death and Apoptosis in NB

toxic against long-term cultured NB cell lines as well as two MDR cell lines more recently isolated from patients after intensive chemotherapy. As most of the cell lines used were wild type for p53, no consistent relationship between p53 status and sensitivity to novel anthracyclines was demonstrated. Finally, WP631 was generally less active against these NB cell lines compared to Dox or O-benzylated anthracyclines. MYCN Overexpression Greatly Enhances Cytotoxicity of WP744

MYC proteins are known to participate in the regulation of apoptosis and cooperativity has been seen between Dox and MYCN for induction of apoptosis in NB [33]. To directly determine whether there was a similar interaction between MYCN expression and sensitivity to WP744, a subclone of the SHEP cell line with conditional, tetracycline-controlled MYCN expression was used [30]. MYCN mRNA was not detectable by Northern analysis in cells treated for one week in medium containing tetracycline (Fig. 2). When cells were washed and re-cultured in the absence of tetracycline for as few as 6 h, abundant MYCN mRNA was seen. Tet 2 (vector only control) and Tet21N cells which harbor the ectopic MYCN construct were grown with or without tetracycline and treated with anthracyclines. Congruent with the findings of Fulda et al., when MYCN expression was high, the sensitivity to Dox was enhanced (IC 50 of 481 nM versus 230 nM; Table 3). Tet21N cells without MYCN expression were more sensitive to WP744 compared to Dox (IC 50 ⫽ 70 nM versus

Dox has been shown to induce apoptosis in susceptible NB cells [6]. Therefore, it seemed plausible that apoptosis might be responsible for cell death seen after exposure to novel anthracyclines as well. To test this hypothesis, apoptosis was assessed by analysis of hypodiploid nuclei as described [34]. SK-N-SH cells demonstrated dose-dependent induction of both apoptosis (Fig. 3) and cell death (Fig. 4) 24 h after exposure to anthracyclines. Moreover, the differences in apoptotic induction between Dox and WP744 were particularly evident at the highest dose of 2000 nM. In fact, both SK-N-SH and SH-SY5Y cells that were treated with Dox at 2000 nM failed to become hypodiploid in contrast to those treated with WP744 at this concentration, yet cell death remained high in these groups (Fig. 4). In contrast, IMR-32 cells demonstrated minimal apoptotic response to treatment with either Dox or WP744, a finding that has previously been reported in response to other chemotherapeutic drugs [35]. This cell line was however quite susceptible to anthracycline-mediated cell death (Fig. 4). Collectively, these data demonstrate that for NB cell lines capable of undergoing apoptosis, WP744 treatment resulted in an enhanced apoptotic response compared to Dox. In IMR-32 cells, which are known to harbor a defective apoptosis pathway, neither drug triggers apoptosis, although cell death occurs in a dosedependent manner. Cytotoxicity of Anthracyclines Is Not Dependent on Apoptosis

The findings above suggested that under certain conditions in at least three cell lines, apoptosis was not

TABLE 3 MYCN Amplification Enhances Anthracycline-Induced Cytotoxicity NB line

Construct

Condition

MYCN expression

n

Dox IC 50

WP744 IC 50

Tet2 Tet2 Tet-21/N Tet-21/N

Vector Vector MYCN MYCN

Media alone Tetracycline Media alone Tetracycline

No No Yes No

3 3 4 4

135 ⫾ 67 159 ⫾ 10 230 ⫾ 33 481 ⫾ 87*

41 ⫾ 16† 53 ⫾ 24† 12 ⫾ 2† 70 ⫾ 26*†

Note. Cells were cultured for 4 days prior to MTT assay. Spectrophotometric data from quadruplicate wells were analyzed to determine IC 50 and the mean ⫾ SD for “n” experiments is shown. Student’s t test was used to determine differences between Dox and WP744. “*” indicates P ⬍ 0.05 compared to cells treated with media alone (no tetracycline). “†” indicates P ⬍ 0.05 compared to cells treated with Dox.

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seen during anthracycline-induced cell death. To more rigorously determine whether caspase-dependent apoptosis was necessary for cell death after anthracycline treatment, NB cells were pre-treated with the broadspectrum caspase inhibitor zVAD-fmk prior to drug exposure. Caspase inhibition resulted in nearly complete abrogation of drug-induced apoptosis (Table 4), confirming that the hypodiploid response was dependent upon caspase activity. Cell death was not affected by zVAD-fmk, demonstrating that inhibition of caspase-driven apoptosis was not sufficient to rescue cells from death after exposure to either anthracycline. In summary, although WP744 treatment more potently and rapidly caused apoptosis in NB cells, other caspase-independent mechanisms are likely responsible for the enhanced cytotoxicity seen with WP744. P-gp Expression in Human NB Cell Lines

Differential cytotoxicity among the various NB cell lines could potentially be explained by differential expression of the plasma membrane drug transporter P-gp. Thus surface phenotyping for expression of this molecule was measured using FCM. The greatest P-gp expression was detected in CHLA-79 cells (Fig. 5), while low-level P-gp expression was found in LA-N-5, CHLA-20, SH-SY5Y, and SK-N-SH cells. No P-gp antigen was detected on IMR-32, SK-N-BE(2), SHEP, and SMS-SAN cells, yet statistically significant differences in cytotoxicity between Dox and O-benzylated anthracyclines were seen in these cell lines (Table 2). Although these data do not exclude a role of P-gp in differential drug sensitivity, they suggest that this is not a major mechanism by which 4=-O-benzylated anthracyclines more potently kill NB.

FIG. 3. Induction of apoptosis in SK-N-SH cells. Cells were incubated in 0, 50, 500, or 2000 nM of either Dox or WP744 for 24 h prior to FACS analysis. Apoptotic cells were estimated by quantifying percent hypodiploid nuclei. Ten thousand nuclei were scanned for each histogram. These data are representative of results obtained in three separate experiments.

FIG. 4. Apoptosis and cell death in response to anthracyclines. NB cells were incubated in 0, 50, 500, or 2000 nM of either Dox or WP744 for 24 h prior to FACS analysis. Apoptotic cells were estimated by quantifying percent hypodiploid nuclei, while cell death was assessed as percent of cells staining with PI from the same treatment group. Ten thousand events were collected for each data point. Data points represent the arithmetic mean while error bars represent SEM for triplicate determinations. “*” indicates that P ⬍ 0.05 for Dox versus WP744. These data are representative of three separate experiments for each cell line.

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TABLE 4 Cell Death After Anthracycline Treatment Is Caspase-Independent

Experiment 1 Control Dox 500 nM WP744 500 nM Experiment 2 Control Dox 500 nM WP744 500 nM

Apop: No zVAD

Apop: 50 ␮M zVAD

Death: No zVAD

Death: 50 ␮M zVAD

6 ⫾ 0.2% 18 ⫾ 1.0% 30 ⫾ 1.1%

4 ⫾ 0.5% 3 ⫾ 0.2% 4 ⫾ 0.2%

30% 59% 81%

29% 61% 82%

4 ⫾ 0.6% 15 ⫾ 0.2% 30 ⫾ 3.1%

3 ⫾ 0.3% 3 ⫾ 0.5% 3 ⫾ 0.19%

34% 46% 64%

39% 53% 66%

Note. SK-N-SH cells were treated for 24 h ⫾ 60 ␮M zVAD-fmk ⫾ Dox or WP744 and apoptosis was measured by FACS analysis of hypodiploid nuclei. Death was quantitated FACS analysis of PI stained whole cells. Apop, apoptosis.

DISCUSSION

Anthracyclines are widely used in the treatment of pediatric and adult solid tumors. A major problem with anthracycline-based therapy is the development of drug resistance. Medicinal chemists have designed many Dox analogs with the intent of enhancing cytotoxicity toward tumor cells, reducing adverse effects of anthracyclines, and bypassing cellular resistance mechanisms. The 4=-O-benzylated Dox analogue WP744 has demonstrated enhanced pro-apoptotic and antitumor activity in a human leukemia model [36]. Our purpose was to determine whether this or other novel anthracyclines manifest greater antitumor activity against NB compared to Dox, and to begin to unravel the mechanism by which the novel agent WP744 affects viability of NB tumor cells. The present study builds on the findings of Faderl [36] and brings to two the number of human tumor types that are more potently killed by this novel anthracycline. Like many adult tumors, NB is usually considered a chemosensitive tumor. Thus, multidrug regimens have proved valuable for the treatment of NB patients. To further improve clinical outcomes, however, we must understand the processes that render tumor cells vulnerable to drug-induced cell death and the factors leading to treatment failure. Treatment failure may be due to either surface protein mediated drug resistance or defects in the apoptotic pathway(s) by which chemotherapeutic agents kill tumor cells. Novel drugs that can bypass such resistance mechanisms may prove very useful clinically. In this study, we selected a panel of 11 wellcharacterized NB cell lines with heterogeneous characteristics and derivation to test the effectiveness of several novel anthracycline drugs. The cell panel included NB lines with and without P-gp expression. It is interesting to point out that the most significant enhancement (⬎6-fold) in cytotoxic activity between Dox and WP744 was seen in SH-SY5Y, SK-N-SH, and CHLA-79 — cell lines which are all positive for

P-gp expression. P-gp expression has been correlated strongly to differentiation status of NB and in fact, very high P-gp expression was also strongly predictive of poor outcome in older children [37, 38]. Others, however, have not confirmed a prognostic value of P-gp for NB [39, 40]. The enhanced cytotoxicity in CHLA-79 cells is perhaps most important, as this P-gp ⫹ cell line was isolated from a patient who had relapsed after myeloablative therapy and bone marrow transplantation. This line has been previously characterized as multidrug resistant and yet, compared to Dox, was significantly more sensitive to WP744. Nonetheless, in this study WP744 was also more potently cytotoxic toward other NB cell lines (SK-N-BE(2), SHEP, and SMS-SANS) that lacked P-gp expression. Thus, it is

FIG. 5. P-gp expression in NB cells. Surface P-gp expression was assessed using FCM. Broken lines represent staining due to the primary antibody (MRK-16) followed by FITC-conjugated secondary antibody. A control group was stained with the FITC-conjugated secondary antibody only (solid lines) to assess nonspecific background fluorescence. These data are representative of similar results obtained in two separate experiments for each cell line.

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likely that other mechanisms in addition to an altered interaction with P-gp account for the enhanced killing effect of WP744 in NB. Depending upon the cellular context and conditions, proteins of the MYC family can play either proapoptotic or anti-apoptotic roles. In neuroblastoma, naturally occurring (endogenous) MYCN expression likely exerts a pathologic anti-apoptotic function for the cells, allowing them to resist apoptosis induced by a variety of stimuli and escape death. This is consistent with the well-accepted notion that reduction of MYC protein expression and its inappropriate expression can be associated with cellular apoptosis [41]. However, in the context of inappropriate, exogenous overexpression of MYCN in the Tet21N cell line, this protein clearly exerts a pro-apoptotic function, enhancing doxorubicin-induced apoptosis [42]. When the role of MYCN expression in sensitivity to WP744 was directly tested in a controlled system, we found that the novel anthracycline WP744 very significantly cooperated with this oncogene for killing of NB cells. This suggests that WP744 may interact with the pro-apoptotic function of MYCN in some NB clones to result in enhanced killing of the tumor cells. While the molecular basis for this interaction is unclear, the finding underscores the potential importance for WP744 as a chemotherapeutic agent, since MYCN amplification is commonly seen in the most aggressive NB in the clinical setting. Whether P-gp expression and/or MYCN status is responsible for the enhanced killing in some the cell lines used in these studies will require further scrutiny. Nonetheless, our aggregate findings with all cell lines suggest that compared to Dox, 4=-O-benzylated anthracyclines more potently kill NB regardless of P-gp or MYCN amplification status. The congruence of the MTT proliferation data between WP744 and WP769 confirms the concept that O-benzylation of Dox confers additional anti-NB cytotoxicity to the anthracycline molecule. In contrast, more complex modifications of the amino sugar, as in WP631, were not associated with enhanced toxicity to NB cell lines. Congruent with previous results demonstrating apoptotic nuclear changes after treatment of NB cells with Dox [6, 43], we found that treatment of two wellcharacterized NB cell lines, SH-SY5Y and SK-N-SH, with Dox and WP744 resulted in the appearance of a distinct hypodiploid nuclear fraction. For Dox, maximal apoptosis was seen with 500 nM of drug for each cell line, while at 2000 nM, cells failed to undergo apoptotic nuclear degradation. In contrast, when these cell lines were treated with 2000 nM WP744, nuclear degradation was similar to that seen with 500 nM. The reason for this discrepancy is not apparent. It is clear, however, that regardless of the magnitude of effect of either anthracycline on NB apoptosis, the drugs killed

the tumor cells very effectively at concentrations exceeding their respective IC 50. Our results failed to show a significant role for caspases in the process by which these anthracyclines kill NB cells. Caspase inhibiting compounds have commonly been used to dissect the pathways of cellular apoptosis and death in response to cytotoxic stimuli, and other investigators have also confirmed that caspases are not necessary for Dox-induced NB death [35, 44]. Although our results clearly confirm that caspase activation is necessary for hypodiploid changes to occur in response to either Dox or WP744, it is similarly clear that these agents, regardless of apoptotic changes, effectively kill NB cells. Some investigators have previously used apoptosis to describe the effectiveness of cytotoxic drugs against cultured NB cells and have elegantly described mechanisms by which chemotherapeutic drugs trigger apoptosis [6, 45]. Our findings, congruent with those of Bian et al. [35], imply that in cultured NB cells, apoptosis and cell death are parallel processes, and cell death may not require caspase-dependent apoptosis to proceed. Anthracyclines exert many effects on cells based on their ability to bind to nuclear DNA, deliver a genotoxic injury, and inhibit topoisomerase 2-mediated DNA repair [46]. Thus, it was unexpected that WP631, a molecule that has many times greater affinity for DNA [47, 48], was not more cytotoxic against the NB cell line panel compared to doxorubicin. This finding would support the hypothesis that the O-benzylated novel analogs exert cytotoxic effects against NB which are not strictly dependent upon DNA binding, but rather, may interact with cell death pathways at other point(s). Indeed, other data from our laboratory (Wu, et al., in press, J. Surg. Res.) suggest that WP744 may more effectively activate NF␬B and Apoptosis Inducing Factor. NF␬B has usually been described as a “prosurvival” molecule, in essence interfering with apoptosis and cell death in many cell types [49, 50]. The opposite has been seen in NB cells however. In SH-SY5Y, NF␬B is stimulated to undergo nuclear translocation and activation by Dox treatment and is responsible for cell death after drug treatment [35]. While the exact interaction between the novel anthracycline WP744 with cell death pathways remains to be elucidated, we believe that this agent may affect other processes in addition to the inhibition of topoisomerase-mediated repair of genotoxic events which is the classic feature of anthracycline cytotoxicity. In conclusion, our analysis of a panel of 11 NB cell lines disclosed enhanced sensitivity to novel O-benzylated anthracyclines compared to Dox. WP744 demonstrated enhanced cytotoxic activity not only for drug-sensitive but also drug-resistant NB cells. This is the first examination of the activity of WP744 against a wide spectrum of well-characterized NB cell lines. Enhanced cytotoxicity in

INGE ET AL.: ENHANCED KILLING OF NEUROBLASTOMA WITH WP744

response WP744 is associated with— but does not require—induction of apoptosis. Our data do not exclude the possibility that enhanced killing in some NB cell lines may be related to P-gp expression; however, this mechanism cannot explain the enhancement of cytotoxic activity in all lines. Perhaps most importantly, these data suggest that novel agents, which were designed to avoid interaction with the P-gp molecule, may well have broadspectrum antitumor activity unrelated to P-gp and may be quite useful in those aggressive NB populations that are MYCN amplified. These drugs may achieve their antitumor activity by bypassing defects in the apoptotic or cell death pathway(s) responsible for the cytotoxic effect of other conventional agents like Dox. High priority should be given to studies examining the exact interaction between these compounds and death pathways in NB cells and other human malignancies and to studies that will establish in vivo effectiveness and toxicology of these novel agents.

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